Ultra‐long‐acting recombinant insulin for the treatment of diabetes mellitus in dogs

Abstract Background For the treatment of diabetes mellitus (DM) in dogs, novel insulins with decreased injection frequency while maintaining safety and efficacy are desirable. Insulin fused with immunoglobulin‐fragment‐crystallizable (Fc) has an ultra‐long plasma half‐life because it recycles through cells, protected from proteolysis. Hypothesis Glycemic control can be achieved in diabetic dogs with a recombinant fusion protein of a synthetic insulin and canine Fc (AKS‐218d) administered subcutaneously once‐weekly. Animals Five client‐owned dogs with naturally occurring DM. Methods Prospective clinical trial in dogs with DM that were recruited from the UC Davis Veterinary Teaching Hospital and local veterinary clinics. Dogs previously controlled using intermediate‐acting insulin q12h were transitioned to once‐weekly injections of a preliminary construct identified as AKS‐218d. The dose of AKS‐218d was titrated weekly for 8 weeks based on clinical response and continuous interstitial glucose monitoring. Clinical signs, body weight, serum fructosamine concentrations, and mean interstitial glucose concentrations (IG) over the preceding week were compared between baseline (before AKS‐218d) and during the last week of treatment. Data were compared using nonparametric paired tests. Results Once‐weekly AKS‐218d, compared to baseline twice‐daily insulin therapy, resulted in no significant changes in clinical signs, median (range) body weight (+0.4 kg [−0.5‐1.1]; P = .6), fructosamine concentration (−75 mmol/L [−215 to +126]; P = .4), or mean IG (+81 mg/dL [−282 to +144]; P = .8). No adverse reactions were reported. Conclusion Control of clinical signs, body weight, and maintenance of glycemia was achieved with this once‐weekly novel insulin construct in 4 of 5 dogs.


| INTRODUCTION
Diabetes mellitus (DM) is a common endocrinopathy in dogs, with an estimated prevalence of 0.3%-0.6%. 1,2 Dogs with DM are invariably insulin-dependent. 3 Treatment of DM in dogs relies upon chronic administration of exogenous subcutaneous (SC) insulin. For many dog owners, the need to administer twice-daily injections adversely affects their quality of life and the perceived well-being of their pet. 4 Owners often cite insulin-related issues as a major cause of anxiety, including worrying about hypoglycemic events and inability to have the dog cared for by others. 4 Faced with the challenges of daily insulin injections, approximately 33% of dog owners elect to euthanize their dog within 1 day of diagnosis. 5 Insulin is a 51-amino acid peptide that tends to form hexamers, especially in the presence of zinc. After injection into the SC tissue, zinc diffuses out of the SC depot, and the hexamers break down into dimers and monomers that then diffuse into the vasculature. 6 To extend the duration of action, traditional insulin formulations typically rely on manipulation of the rate of hexamer dissociation in the SC depot to slow insulin absorption into the blood. 6 Furthermore, decreasing the affinity of insulin to its receptor also prolongs duration of action. 7 With manipulation of absorption rate and receptor affinity, currently available insulin formulations have time-action profiles suitable for use as onceor twice-daily SC injections. 8 We present data on a novel ultra-long-acting insulin construct (AKS-218d) intended for once-weekly administration in diabetic dogs.
The active molecule in this formulation is a fusion protein of synthetic insulin and the canine fragment crystallizable (Fc) region of immunoglobulins. This fusion protein is designed to be nonimmunogenic is a ligand to the insulin receptor, but also binds to the host neonatal Fc receptor (FcRn). Binding to the FcRn leads to recycling of the insulin fusion molecule intracellularly, which extends its half-life compared to native insulin. 9 Preliminary data from healthy laboratory dogs (US patent US10961294) suggest that the half-life of this molecule would allow for once-weekly administration with prolonged glucoselowering effect. We hypothesized that in dogs with naturally occurring DM, this Fc-insulin fusion protein would control clinical signs, body weight, and blood glucose concentrations (BG) with once-weekly injections. This was a dose-escalation study, with the aim of assessing the ability of once-weekly AKS-218d injections to maintain or improve glycemic control in diabetic dogs previously treated using conventional twice-daily insulin administration.

| MATERIALS AND METHODS
Dogs were recruited to this study from the University of California, Davis Veterinary Medical Teaching Hospital and local veterinary clinics. Dogs of any age between 3 and 30 kg with no recent changes in body weight (defined as <5% in the past 3 months) were included if they were diagnosed with naturally occurring DM (in accordance with the Project Agreeing Language in Veterinary Endocrinology [ALIVE] definition for DM diagnosis). 10 Dogs were required to have been treated with any insulin formulation (≤1.5 U/kg per injection) for at least 2 months before enrollment. Dogs were required to have moderate to good glycemic control as defined by mild clinical signs of DM (subjectively reported by dog owners) and stable serum fructosamine concentrations (defined as 2 serum fructosamine concentrations measured at least 3 weeks apart that were within 100 mmol/L of each other). After inclusion, the degree of glycemic control on prestudy insulin was also assessed by measuring interstitial glucose concentration (IG) continuously (see below) during the first week (Figures 1 and   2). Additionally, dogs must have been fed a consistent diet throughout the study period. Exclusion criteria included any concurrent illness that might preclude following the dog >6 months and that might affect insulin requirements, poorly controlled hypothyroidism, positive urine culture, inability to tolerate the flash glucose monitor (see below) and with a history of diabetic ketoacidosis within the past 2 months, or with a history of diabetic ketoacidosis within the past 2 months. A flash glucose monitoring system (FGMS; FreeStyle Libre, Abbott Laboratories, Illinois) provides clinically accurate estimates of glucose in diabetic dogs and therefore was used throughout the study. 11 A FGMS sensor was applied to the skin on the dorsolateral lumbar area as previously described. 9 This FGMS measures IG once per minute and records 15-minute IG averages for up to 14 days.
Minute-by-minute measurements can be obtained by scanning the sensor more frequently. After this initial screening visit, dogs were discharged for 10-14 days to continue receiving their prestudy insulin twice daily and to ensure compliance with the FGMS.
On visit number 2 (day 0), thoracic radiography and abdominal sonography were performed. If no exclusion criteria were met, a new FGMS sensor was applied and the dog received the first injection of AKS-218d at approximately 1.5 nmol/kg SC. This dose was chosen based on preliminary data from healthy purpose-bred dogs with the intention of approximating a 0.3 U/kg/day dose of conventional insulin. Based on preliminary data from healthy purpose-bred dogs, it was anticipated that hypoglycemia would be unlikely at this dose, that escalation of the AKS-218d dose over the succeeding weeks would be required to maintain glycemic control, and that maximal AKS-218d effects were expected to peak within the first 24-36 hours after injection. Because the potency of AKS-218d was not known in dogs with naturally occurring DM, and in order to minimize the risk of hypoglycemia, dogs were hospitalized for monitoring and hourly FGMS scans were conducted for 48 hours after the first injection. If no hypoglycemia was observed, dogs were discharged from the hospital after 48 hours of observation; if hypoglycemia leading to clinical signs was observed, IV dextrose supplementation would be administered at the discretion of the study investigators. At home, the need for any additional glycemic control between weekly AKS-218d doses was assessed by the study investigators in conjunction with the owner.
Supplemental insulin was not administered by the owner unless instructed otherwise by the study investigators; when deemed necessary, based on clinical signs including excessive polyuria, polydipsia, and polyphagia in conjunction with FGMS data indicating persistent hyperglycemia, owners were instructed to also administer prestudy insulin at half the prestudy dose to address any hyperglycemic events.
After discharge from the hospital, dogs were re-evaluated weekly via a full physical examination including body weight, serum samples were collected for drug concentration and anti-drug antibodies (ADA), the FGMS sensor was replaced, and AKS-218d was administered SC. The dose of AKS-218d was adjusted weekly as necessary based on the past week's IG concentrations, body weight, and clinical response (serum drug concentration data were not available to the study investigators during the study). In total, dogs were treated with AKS-218d 8 times (ie, for 56 days). During the 5th visit (end of week 4) and the final visit (1 week after the last injection), samples were collected for hematology, serum biochemistry, serum fructosamine concentration, urinalysis and urine protein: creatinine ratio via cystocentesis to survey for potential adverse drug effects.

| Measurement of serum concentrations of AKS-218D and anti-drug antibodies
Serum drug concentrations of AKS-218d and ADA were measured via sandwich ELISAs specifically developed by Akston Biosciences (Beverly, Massachusetts) for the purpose of this study. To measure concentrations of AKS-218d, microtiter plates were coated with purified anti-insulin antibodies to capture the AKS-218d therapeutic molecules in the serum samples. The captured serum AKS-218d then was quantitated using a goat anti-dog IgG-Fc-horseradish peroxidase (HRP) detection antibody followed by a tetramethylbenzidine substrate system. The tetramethylbenzidine substrate changes color when it reacts with the HRP that is conjugated to the detection antibody. The enzyme substrate reaction was stopped by the addition of a stop reagent (1% H 2 SO 4 ) and the color intensity was measured in a microplate reader at 450 nm.   variability percentage (GVP), which was calculated as previously described. 12 In brief, the GVP method calculates the length of IG line from continuous data by using a trigonometric analysis of the data and is used as a superior indicator of intraday BG variability. 12

| RESULTS
Six dogs were enrolled into the study.  adverse events (except for infrequently low IG). Glycemic control was similar between the once-weekly and twice-daily protocols suggesting that the novel therapy will offer considerable benefits to the dog and pet owner.
In people, increasing dosing frequency of any drug is associated with decreased adherence to treatment protocol. 13 In diabetic people, poor adherence is compounded by the pain of insulin injection. 14,15 Human patients consider insulin injections to be a serious burden with a negative impact on quality of life. 14 Although adherence has not been studied in people giving insulin injections to dogs, it is anticipated that the problems noted in human patients will be compounded by the physical and emotional challenges of performing repeated injections in dogs. It is also unknown how the act of injection itself affects compliance and the owners' short and long-term decisions to treat. Although not exclusively associated with these problems, it is reasonable to assume that the high euthanasia rate (30%-40%) soon after DM diagnosis is associated with client's perceived compliance issues, especially when considering these results in a population of dogs covered by pet medical insurance. 4,5 A treatment that offers an alternative to daily injections should minimize these barriers to greater treatment success, improve quality of life for the pet and owner, and increase dog survival. In this context, it is also important to consider not just the treatment choice per se, but also how it affects monitoring intensity and cost.
Because ours was the first study to assess the efficacy of this novel ultra-long-acting insulin construct in diabetic dogs, we only recruited dogs that were well-controlled, with the intention of comparing the treatment outcomes of the insulin-Fc fusion protein to that of the standard insulin formulations. We did not include dogs that were newly diagnosed with DM because of an initial perception that glycemic control might take longer than usual to achieve. Considering the long duration of action of this construct and the potential for longterm hypoglycemia in the event of an overdose, a conservative starting dose and slow rate of dose increase was advised. Although our data set is limited, it seems that a lag of about a week until full response led us to escalate the dose more then was necessary, leading to a peak at week 3 and a dose correction thereafter. Using the data from this study, it is expected that future dose exploration studies would generate a starting dose that both controls clinical signs sufficiently and avoids hypoglycemia in all dogs.
Anti-drug antibody (ADA) formation is a common sequela of insulin therapy in dogs, cats, and humans, whether they are treated with heterologous or autologous insulin. [16][17][18] The clinical importance of these antibodies is unclear, but they generally do not cause clinical problems. 8,17,18 Here, ADA developed to AKS-218d in 1 out of 5 dogs beginning about 6 weeks after starting treatment, which corresponded to a decrease in measured serum concentration of the study drug and led to the recurrence of clinical signs of diabetes. The development of these antibodies did not impede response to porcine insulin after the study ended. It remains to be established whether this antibody response was unique to the study drug construct or to administration of a heterologous insulin. During the development of the Fc-insulin fusion construct used in this study, many Fc-insulin fusion molecules containing different mutated insulin peptides were screened for manufacturability, bioactivity, and immunogenicity. 19 For example, attributes like protein glycosylation can greatly influence uptake and proteolytic processing of antigens and has been implicated in many aspects of adaptive immune activation. Therefore, in the development of an insulin-Fc protein construct, the combination of stability, high affinity and potency and long half-life, combined with safety and insulin activity, have to be assessed and the optimal construct identified.
Considering the long duration of action of this novel insulin con- In most insulin formulations, retardation of absorption from the SC depot relies on the formation of insulin hexamers, a process that is a function of insulin concentration and the precise ratio of insulin to other molecules (such as zinc, protamine, or m-cresol) and local tissue pH. 6 This markedly limits the ability to vary the concentrations of insulin formulations and is the reason why dilute formulations have failed to maintain prolonged duration of action in the past. 23 Because the prolonged duration of action of AKS-218d does not rely on slowing absorption of insulin from the SC tissue, it can be formulated in any concentration, making it exceedingly convenient for use in small dogs.
An insulin that leads to lesser between-day variability likely would require simpler monitoring protocols compared to formulations that are associated with more variability. One of the major factors contributing to between-day variability of insulin dosage is erratic absorption of insulin crystals that vary in size and shape. 24,25 In contrast, the novel insulin construct AKS-218d does not precipitate in the SC tissue. That, together with the fact that AKS-218d likely achieves steady state after a few weeks of treatment (exemplified by the fact that the AKS-218d dose remained fairly constant throughout the last 5 weeks of the study), suggested that there would be a decreased between-day variability in the last week of the study compared to baseline. We did not observe that positive outcome, possibly because of lack of statistical power.
Interstitial glucose concentrations were measured continuously throughout the study, but these data were used primarily to aid in dose adjustments and only secondarily as outcome measures. Neither the optimal target IG nor the optimal BG in dogs is known, such that diabetic control is defined instead as "the absence of clinical signs and hypoglycemia." 8 In addition, although more data are available regarding BG targets, in this study we measured IG believing that it would better reflect tissue physiologic requirements for insulin and a broader perspective on trends and not just single time points. 26 Use of a continuous glucose monitoring system (CGM) in this study of a novel ultra-longacting insulin construct was believed to be important in the context of identifying subclinical hypoglycemic events. The CGM used in our study has been validated for use in dogs, showing excellent correlation between BG and IG overall. 11 However, like other glucometers intended for use in people, this CGM underestimates glucose concentrations in the hypoglycemic range (by about 20 mg/dL). 11 This possibly means that the frequency of low IG events in our study may be an overestimation of the frequency of true hypoglycemic events, potentially explaining why we did not observe clinical hypoglycemia. In the future, it will be important to establish a dosing scheme and develop appropriate monitoring protocols that will optimize the use of once-weekly insulin formulations. The current study did not detect a difference in the frequency of low IG readings between q12h insulin at baseline and once-weekly treatment with AKS-218d at the end of the study. It is plausible that a clinically relevant difference was not detected because of the low power of this study.
In summary, once-weekly administration of AKS-218d was sufficient for maintaining body weight, stable serum fructosamine concentrations, and controlling clinical signs in these 5 dogs with naturally occurring diabetes mellitus.

ACKNOWLEDGMENT
Funding provided by Akston Biosciences, Inc, grant X0VC41. We would like to thank Timmy and his family, for accepting the challenge of being first and maintaining a positive attitude, even when times got rough.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.